Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. The current intensified chemotherapy is associated with high response rates, but also with serious short and long-term side effects in these young patients. KPT-8602 is a second generation exportin-1 (XPO-1) inhibitor with potent activity against ALL in pre-clinical models and with minimal effects on normal cells.
In this study, we evaluated the possible synergy between the new KPT-8602 drug and currently used drugs such as dexamethasone, vincristine and doxorubicin for the treatment of B and T-ALL. We further searched to elucidate the mechanism behind the synergistic interaction.
T-ALL cell lines DND-41 and SUP-T1, and B-ALL cell lines 697 and BV-173 were used. Treatment with dexamethasone (DEXA), KPT-8602, doxorubicin and vincristine or combinations was performed for 24 h or 48 h depending on the experiment. Viability was measured via ATP-lite and apoptosis via AnnexinV-PI staining. Gene expression was assessed via RT-qPCR and using the NanoString nCounter PanCancer pathways panel.
ChIP-sequencing was performed by cross-linking proteins on the DNA, followed by nuclei isolation and chromatin fragmentation. Overnight chromatin immunoprecipitation was performed in the presence of antibodies (Glucocorticoid receptor, H3K4me3, H3K27Ac). Illumina Hiseq 2000 was used for analysis. Raw sequencing data were mapped to the human reference genome (GRCh37/h19) using Bowtie.
Human leukemic mononuclear cells were injected through tail vein injection in NSG mice. Disease was monitored by peripheral blood withdrawal and staining with human CD45. Moribund mice were sacrificed and leukemic cells harvested from the spleen to transduced ex vivo with a GFP-fLuc lentiviral vector. After multiple engraftment/sorting rounds, luc+ samples were injected into a larger cohort of NSG mice for in vivo treatment. Once engraftment was established, treatment started and disease evolution was followed via bioluminescent imaging. KPT-8602 or vehicle (DMSO) was given via gavage for 5 days, followed by 2 days off. DEXA was given via the water (3 days on, 2 days off). After two weeks of treatment, mice were sacrificed and leukemic infiltration assessed.
Of all combinations tested, KPT-8602 together with DEXA showed the strongest synergistic effect in human B- and T-ALL cell lines. This synergy was confirmed in vivo on one B-ALL and two T-ALL PDX, where KPT-8602 and DEXA together led to a significant reduced leukemic burden compared to single treatments. Combination treatment reduced leukemic infiltration in the blood, spleen and bone marrow and led to a prolonged leukemia-free survival (p < 0.0001).
Mechanistically, the KPT-8602 and DEXA combination caused increased apoptosis, reduced cell cycle gene expression and led to histone depletion. mRNA expression analysis and ChIP-seq revealed that addition of KPT-8602 to DEXA increased glucocorticoid receptor (GR) expression and its binding to GRE regulatory DNA regions. Moreover, GR target genes such as NFKBIA, PER1 and STAG3 showed increased expression upon combination treatment compared to single drug treatment. DEXA concentrations could be reduced 5 to 10-fold in the presence of KPT-8602 to achieve the same level of NFKBIA and GR induction as with DEXA alone.
Our pre-clinical study demonstrates that combination of KPT-8602 and dexamethasone is highly synergistic in vivo. This opens the possibility to reduce dexamethasone concentrations and hence reduce side effects in future clinical trials.